Coronaviruses — Drug Discovery and Therapeutic Options

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Coronaviruses — drug discovery and therapeutic options

Alimuddin Zumla1*, Jasper F. W. Chan2*, Esam I. Azhar3*, David S. C. Hui4* and Kwok‑Yung Yuen2* Abstract | In humans, infections with the human coronavirus (HCoV) strains HCoV‑229E, HCoV‑OC43, HCoV‑NL63 and HCoV‑HKU1 usually result in mild, self-limiting upper respiratory tract infections, such as the common cold. By contrast, the CoVs responsible for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), which were discovered in Hong Kong, China, in 2003, and in Saudi Arabia in 2012, respectively, have received global attention over the past 12 years owing to their ability to cause community and health-care-associated outbreaks of severe infections in human populations. These two viruses pose major challenges to clinical management because there are no specific antiviral drugs available. In this Review, we summarize the epidemiology, virology, clinical features and current treatment strategies of SARS and MERS, and discuss the discovery and development of new virus-based and host-based therapeutic options for CoV infections.

Coronaviruses (CoVs; subfamily Coronavirinae, family SARS in Hong Kong and other regions10,11,18–21. The high Coronaviridae, order Nidovirales) are a group of highly infectivity of SARS was highlighted by the super-spread- diverse, enveloped, positive-sense, single-stranded ing event at a major teaching hospital in Hong Kong in RNA viruses that cause respiratory, enteric, hepatic which 138 people, including many previously healthy and neurological diseases of varying severity in a broad health-care workers, were infected within 2 weeks of range of animal species, including humans1–3. CoVs exposure to an index patient who was being managed in are subdivided into four genera: Alphacoronavirus, a general medical ward for community-acquired pneu- Betacoronavirus (βCoV), Gammacoronavirus and monia10,22. Through international air travel, SARS-CoV Deltacoronavirus2–7. Over the past 12 years, two novel was spread to 29 countries and regions with a total of βCoVs, severe acute respiratory syndrome CoV (SARS- 8,098 cases and 774 fatalities (9.6% of cases) by the end CoV) and Middle East respiratory syndrome CoV of the epidemic in July 2003 (REF. 23) (see Supplementary (MERS-CoV), have emerged, and these viruses can information S1 (figure, parts a,b)). cause severe human diseases8,9. The lack of effective drug A retrospective serological survey suggested that treatment and associated high morbidity and mortality cross-species transmission of SARS-CoV or its variants rates of these two CoVs as well as their potential to cause from animal species to humans might have occurred fre- epidemics highlight the need for novel drug discovery quently in the wet market, where high seroprevalence for the treatment of CoV infections. was detected among asymptomatic animal handlers24. A close variant of SARS-CoV was isolated in palm civets Epidemiology of SARS and MERS in Dongmen market, Shenzhen, China, in 2003 (REF. 25). SARS. SARS-CoV emerged first in southern China During the small-scale SARS outbreaks in late 2003 and and rapidly spread around the globe in 2002–2003 early 2004, three of the four patients had direct or indi- (REFS 8,10,11). In November 2002, an unusual epidemic rect contact with palm civets26,27. However, viral genetic of atypical pneumonia with a high rate of nosocomial sequence analysis demonstrated that the SARS-CoV- transmission to health-care workers occurred in Foshan, like virus had not been circulating among masked palm 12,13 *All authors contributed Guangdong, China . In March 2003, a novel CoV civets in markets for a long time, and a serological study equally to this work. was confirmed to be the causative agent for SARS, and was showed that only caged market civets and not wild civets 14–17 28 Correspondence to K.-Y.Y. thus named SARS-CoV . A 64‑year-old nephrologist were infected with the SARS-CoV-like virus . CoVs that [email protected] who travelled from southern China to Hong Kong on are highly similar to SARS-CoV have been isolated from doi:10.1038/nrd.2015.37 21 February 2003 became the index case of subsequent Chinese horseshoe bats since 2005 (REFS 29–32). These Published online 12 Feb 2016 large community and health-care-associated outbreaks of SARS-like CoVs from bats share 88–95% sequence

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Zoonotic virus homology with human or civet CoV isolates, which of transmission and other potential sources of MERS- A virus that can transmit suggests that bats were probably the natural reservoir of CoV for optimization of treatment and prevention an infectious disease from a close ancestor of SARS-CoV4,33,34. strategies for MERS56,57. animals (usually vertebrates) to humans. MERS. The isolation of a novel βCoV from a patient Clinical features of SARS and MERS. Patients with SARS in Jeddah, Saudi Arabia, who died of severe pneumonia or MERS present with various clinical features, ranging and multi-organ failure in June 2012, was first reported from asymptomatic or mild respiratory illness to fulmi- in September 2012 (REF. 35). Initially named ‘human nant severe acute respiratory disease with extrapulmo- coronavirus Erasmus Medical Center’, the virus was nary manifestations8,9. Both diseases have predominantly later renamed MERS-CoV by international consensus, respiratory manifestations, but extrapulmonary features and the disease was called Middle East respiratory syn- may occur in severe cases56 (see Supplementary infor- drome (MERS)36. Retrospective analysis of a cluster of mation S2 (table)). Notably, early treatment is especially nosocomial cases in April 2012 in Jordan confirmed that important for patients with severe MERS because this MERS-CoV was also responsible for that outbreak37. disease progresses to respiratory distress, renal failure Over the past 3 years, MERS-CoV has continued to and death much more rapidly than SARS does. The spread within and beyond the Middle East, and there three- to four-fold higher case-fatality rate of MERS rel- are ongoing reports of sporadic cases and community ative to SARS may be related to the higher median age and health-care-associated clusters of infected individu- and prevalence of comorbidities in patients with MERS als in the Middle East, especially in Saudi Arabia and the as well as the different pathogenesis of the two dis- United Arab Emirates9,38. Travel-related cases and clus- eases9,58–61. Comorbidities associated with severe MERS ters have also been increasingly reported on other include obesity, diabetes mellitus, systemic immuno- continents9. As of 9 October 2015, 1,593 laboratory- compromising conditions and chronic cardiac and pul- confirmed cases of MERS, including 568 deaths, have monary diseases9,60,62,63. Although the rate of secondary been reported to the World Health Organization39 (see transmission among household contacts of index MERS Supplementary information S1 (figure, parts c,d)). patients (which is approximately 4%) and the estimated MERS-CoV is considered primarily to be a zoonotic pandemic potential of MERS-CoV are lower than those virus that has the capability of non-sustained person- of SARS-CoV, the rapidly progressive clinical course and to‑person spread9. Serological and virological studies high fatality of MERS continues to pose a major threat to have shown that camels and bats are the most likely at-risk populations64–71 (see Supplementary information animal reservoirs of MERS-CoV9,40–47. Although not all S2 (table)). primary cases of MERS were individuals who had direct contact with camels, such exposure is considered to be Current management strategies for SARS and MERS. an important factor for the spread of MERS-CoV, as Supportive care — including organ support and preven- evidenced by the substantially increased seroprevalence tion of complications, especially acute respiratory dis- of anti-MERS-CoV antibodies among individuals tress syndrome, organ failure and secondary nosocomial with occupational exposure to camels, such as camel infections — remains the most important management shepherds and slaughterhouse workers, relative to strategy for SARS and MERS, as there is currently no the general population in Saudi Arabia48,49. Person- specific antiviral treatment that has been proven to to‑person transmission of MERS-CoV has occurred be effective in randomized controlled trials8,9,56,72–75. in health-care facilities and family clusters50–53. The Numerous compounds have been found to inhibit the recent, large health-care-associated outbreaks in Jeddah entry and/or replication of SARS-CoV and MERS-CoV and South Korea have been attributed to poor com- in cell culture or in animal models, but activity in vitro pliance with infection control measures54,55. Further and even in animal experiments does not necessarily studies are needed to fully understand the exact mode translate into efficacy in humans8,9. Owing to the high morbidity and mortality rates of SARS and MERS, some of these antiviral drugs and immunomodulators Author addresses have been used empirically or evaluated in uncontrolled 8,10,21,72–90 1Division of Infection and Immunity, University College London, and NIHR Biomedical trials (TABLE 1). Substantial efforts are underway Research Centre, UCL Hospitals NHS Foundation Trust, 307 Euston Road, London to discover new therapeutic agents for CoV infections NW1 3AD, UK. and these investigations are based on our understanding 2State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, of the basic virology of CoVs. Importantly, treatment Research Centre of Infection and Immunology, Department of Microbiology, with these investigational therapies requires appli- University Pathology Building, Queen Mary Hospital, The University of Hong Kong, cation of standard research treatment protocols and 102 Pokfulam Road, Pokfulam, Hong Kong Special Administrative Region of the systematic clinical and virological data collection in People’s Republic of China. controlled research trials, with the approval of the local 3Special Infectious Agents Unit, King Fahd Medical Research Centre, and Medical Laboratory Technology Department, Faculty of Applied Medical Sciences, King ethics committee. Abdulaziz University, P.O. Box 128442, Jeddah — 21362, Kingdom of Saudi Arabia. 4Division of Respiratory Medicine and Stanley Ho Centre for Emerging Infectious Development of anti-CoV therapeutics Diseases, The Chinese University of Hong Kong, Prince of Wales Hospital, 30–32 Ngan Key CoV targets for new drug development. Despite Shing Street, Shatin, New Territories, Hong Kong Special Administrative Region of their high species diversity, CoVs share key genomic the People’s Republic of China. elements that are essential for the design of therapeutic

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Table 1 | Therapeutic interventions used in patients with SARS and MERS Type of Therapeutic Treatment effects Refs intervention intervention Treatments used for SARS patients Antivirals Ribavirin No significant effect on clinical outcome 10,21 Ribavirin, Patients who received ribavirin, lopinavir–ritonavir and a 76,77 lopinavir–ritonavir + corticosteroid had lower 21‑day ARDS and death rates than corticosteroids those who received ribavirin and a corticosteroid Interferon Interferon Associated with improved oxygen saturation and more rapid 78 combination alfa‑1 + corticosteroid resolution of radiographic lung opacities than systemic corticosteroid alone (uncontrolled study) Corticosteroids Pulsed Associated with an increased 30‑day mortality rate (adjusted 79–81 methylprednisolone OR = 26.0, 95% CI = 4.4–154.8). Disseminated fungal infection and avascular osteonecrosis occurred following prolonged systemic corticosteroid therapy A randomized, placebo-controlled study showed that plasma 82 SARS-CoV RNA levels in weeks 2–3 of the illness were higher in patients given hydrocortisone (n = 10) than those given normal saline (n = 7) in the early phase of the illness, suggesting that early use of pulsed methylprednisolone might prolong viraemia Convalescent-phase Convalescent-phase Has been used for severe respiratory tract infections 85,272, plasma plasma therapy including SARS and influenza. A systematic review and 273 exploratory meta-analysis of patients with SARS or influenza treated with convalescent-phase plasma showed a reduction in mortality, but the treatment success was determined by its availability and timely administration Among 80 non-randomized SARS patients who were given 83,84 convalescent-phase plasma, the discharge rate at day 22 was 58.3% for patients (n = 48) treated within 14 days of illness onset versus 15.6% for those (n = 32) treated beyond 14 days Treatments used for MERS patients Combination of Ribavirin + interferon No significant effect on clinical outcome; case–control study 86–89 antivirals and alfa‑2a or interferon showed significantly improved survival (14 out of 20 and 7 interferons alfa‑2b out of 24 in the treated and control groups, respectively; P = 0.004) at 14 days, but not at 28 days Ribavirin + interferon Retrospective analyses showed no significant effect on 89 beta‑1a clinical outcome Ribavirin, lopinavir– Viraemia resolved 2 days after commencement of treatment 90 ritonavir + interferon in a patient with severe MERS alfa‑2a Corticosteroids Pulsed Patients with severe MERS who were treated with systemic 87,88, methylprednisolone corticosteroid with or without antivirals and interferons had 274 no favourable response ARDS, acute respiratory distress syndrome; CI, confidence interval; CoV, coronavirus; MERS, Middle East respiratory syndrome; OR, odds ratio; SARS, severe acute respiratory syndrome.

targets (FIG. 1). The large replicase polyprotein 1a (pp1a) and carboxy-terminal membrane fusion S2 subunits. and pp1ab, which are encoded by the 5ʹ‑terminal open Cleavage at the protease site at the S1–S2 junction is Spike glycoprotein (S) reading frame 1a/b (ORF1a/b), are cleaved by two viral required to activate membrane fusion, virus entry and 9 A major immunogenic antigen proteases, the papain-like protease (PLpro) and the syncytium formation . Binding of the S1 subunit recep- of coronaviruses that 3C‑like protease (3CLpro), to produce non-structural tor-binding domain (RBD) to the host receptor triggers is essential for interactions proteins (NSPs) such as RNA-dependent RNA poly conformational changes in the S2 subunit (the stalk between a virus and host cell receptor, and is an important merase (RdRp) and helicase, which are involved in region of S) to bring the viral and cell membranes into 9,91 92 therapeutic target. the transcription and replication of the virus (FIG. 2). close proximity and enable fusion . Monoclonal anti- Numerous enzyme inhibitors targeting these proteins bodies (mAbs) against the S1 subunit RBD and fusion Syncytium have shown anti-CoV activity in vitro. inhibitors targeting the S2 subunit have potent anti-CoV A multinucleated cell resulting The surface structural spike glycoprotein (S) is of par- activity in vitro and/or in vivo92–100. The key functional from the fusion of the host membranes of neighbouring ticular interest for antiviral development because of its host receptors utilized by human pathogenic CoVs cells infected by various critical role in the virus–cell receptor interaction. S is include angiotensin-converting enzyme 2 (ACE2; used by viruses, including CoVs. composed of the amino-terminal receptor-binding S1 SARS-CoV and human CoV (HCoV)-NL63), dipeptidyl

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Non-structural proteins Structural and accessory proteins

1 5,000 10,000 15,000 20,000 25,000 30,000 32,500

SARS-CoV ORF1a ORF1b 5ʹ 3ʹ

PLpro 3CLpro RdRp Hel S E M N

MERS-CoV ORF1a ORF1b 5ʹ 3ʹ S E M N PLpro 3CLpro RdRp Hel

• Proteolysis • Proteolysis • Viral replication • Viral N • Interferon and transcription replication antagonist • Virion assembly • Deubiquitination • Interferon antagonist (SARS-CoV) • DeISGylation • Viral suppressor of RNA silencing (SARS-CoV) M • Virion assembly • Interferon antagonist (MERS-CoV) E • Virion assembly • Viroporin (SARS-CoV) S • Virus–cell receptor binding

Figure 1 | Genomes and structures of SARS-CoV and MERS-CoV. The typical coronavirus (CoV) genome is a single-stranded, non-segmented RNA genome, which is approximately 26–32 kb. It contains 5ʹ‑methylated caps and 3 ‑polyadenylated tails and is arranged in the order of 5 , replicase genes, genes encoding structural proteins ʹ ʹ Nature Reviews | Drug Discovery (spike glycoprotein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N)), polyadenylated tail and then the 3ʹ end. The partially overlapping 5ʹ‑terminal open reading frame 1a/b (ORF1a/b) is within the 5ʹ two-thirds of the CoV genome and encodes the large replicase polyprotein 1a (pp1a) and pp1ab. These polyproteins are cleaved by papain-like cysteine protease (PLpro) and 3C‑like serine protease (3CLpro) to produce non-structural proteins, including RNA-dependent RNA polymerase (RdRp) and helicase (Hel), which are important enzymes involved in the transcription and replication of CoVs. The 3ʹ one-third of the CoV genome encodes the structural proteins (S, E, M and N), which are essential for virus–cell-receptor binding and virion assembly, and other non-structural proteins and accessory proteins that may have immunomodulatory effects297. Particle image from REF. 296, Nature Publishing Group. MERS, Middle East respiratory syndrome; SARS, severe acute respiratory syndrome.

peptidase 4 (DPP4; used by MERS-CoV), aminopepti- abrogate this proteolytic cleavage and partially block dase N (used by HCoV‑229E), and O‑acetylated sialic cell entry109. MERS-CoV S is also activated by furin, a acid (used by HCoV‑OC43 and HCoV‑HKU1)101–106. serine endoprotease that has been implicated in the pro- The host receptor is important in determining the path- cessing of fusion proteins and cell entry of other RNA ogenicity, tissue tropism and host range of the virus. viruses, including HIV, avian influenza A/H5N1 virus, mAbs or agents that target the host receptor are poten- Ebola virus, Marburg virus and flaviviruses110. Furin tial anti-CoV agents so long as they do not induce is also involved in MERS-CoV S1/S2 cleavage during immunopathological effects in animal models. egress from the infected cell110. Monotherapy and/or CoVs enter the host cell using the endosomal pathway combinatorial treatment with inhibitors of host pro- and/or the cell surface non-endosomal pathway9 (FIG. 2). teases involved in the various cell entry pathways have Low pH and the pH‑dependent endosomal cysteine potent anti-CoV activity in vitro and should be further protease cathepsins help to overcome the energetically evaluated in animal studies109,111. unfavourable membrane fusion reaction and facilitate CoVs disassemble inside the host cell and release endosomal cell entry of CoVs107,108. Other host proteases, the nucleocapsid and viral RNA into the cytoplasm, such as transmembrane protease serine 2 (TMPRSS2) after which ORF1a/b is translated into pp1a and and TMPRSS11D (also known as airway trypsin-like pp1ab, and the genomic RNA is replicated91. The protease), cleave S into the S1 and S2 subunits to acti- numerous NSPs produced by the cleavage of pp1a vate S for cell surface non-endosomal virus entry at the and pp1ab form the replication–transcription com- plasma membrane109. Inhibitors of these proteases can plex. Attachment of the hydrophobic domains of the

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CoV replication–transcription complex to the limiting sterol metabolism, protein processing and DNA synthesis membrane derived from the endoplasmic reticulum or repair122–127. The major disadvantage of this approach produces typical CoV replication structures includ- is that although many of the identified drugs exhibit ing double-membrane vesicles (DMVs) and convo- anti-CoV activities in vitro, most are not clinically useful luted membranes112,113. Novel agents, such as K22, that because they are either associated with immunosuppres- target membrane-bound CoV RNA synthesis inhibit sive effects or they have anti-CoV half-maximal effective

DMV formation of a broad range of human and ani- concentration (EC50) values that markedly exceed the 112 mal CoVs . The full-length positive strand of genomic peak serum concentration (Cmax) levels that are achievable RNA is transcribed to form a full-length negative-strand at therapeutic dosages. A notable exception, which was template for the synthesis of new genomic RNAs and found to be effective in a non-human primate model and overlapping subgenomic negative-strand templates9,91. in non-randomized clinical trials, is the anti-HIV protease Subgenomic mRNAs are then synthesized and translated inhibitor lopinavir–ritonavir76,77,128 (TABLE 1). to produce the structural and accessory proteins9,91. The The third approach for anti-CoV drug discovery helical nucleocapsid, formed by the assembly of nucleo involves the de novo development of novel, specific agents capsid protein (N) and genomic RNA, then interacts based on the genomic and biophysical understanding of with S, envelope protein (E), and membrane protein the individual CoVs. Examples include siRNA molecules (M) to form the assembled virion9. The virion is released or inhibitors that target specific viral enzymes involved into the extracellular compartment by exocytosis and in the viral replication cycle, mAbs that target the host the viral replication cycle is repeated9. Small interfering receptor, inhibitors of host cellular proteases, inhibitors of RNAs (siRNAs) targeting these structural genes could virus endocytosis by the host cell, human or humanized be useful in the treatment of CoV infections, and fur- mAbs that target the S1 subunit RBD and antiviral pep- ther optimization of the in vivo delivery of siRNAs may tides that target the S2 subunit (FIG. 2). Although most of enable their clinical use. these drugs have potent in vitro and/or in vivo anti-CoV activity, their pharmacokinetic and pharmacodynamic Approaches to anti-CoV drug screening. The only two properties and side-effect profiles have yet to be evaluated human-pathogenic CoVs known before the SARS epi- in animal and human trials. Furthermore, the develop- demic were HCoV‑229E and HCoV‑OC43, which ment of these candidate drugs into clinically useful ther- usually cause self-limiting upper respiratory tract infec- apeutic options with reliable delivery modes for patients tions2. Therefore, researchers and research facilities, usually takes years. especially those involved in antiviral development, were Overall, these three drug discovery approaches are underprepared when SARS-CoV suddenly emerged in often used together during emerging CoV outbreaks to 2003. Subsequently, three general approaches were used identify candidate drug compounds that can be broadly to discover potential anti-CoV treatment options for classified into virus-based and host-based treatment human-pathogenic CoVs — especially SARS-CoV and options. the emerging MERS-CoV — that are associated with more severe disease than the other HCoVs are9,114,115. Virus-based anti-CoV treatment options The first approach to drug discovery is to test exist- Viral nucleosides, nucleotides and nucleic acids. ing broad-spectrum antiviral drugs that have been used Nucleosides and nucleotides are the building blocks of to treat other viral infections by using standard assays viral nucleic acids (FIG. 2). Drugs that target nucleosides that measure the effects of these drugs on the cytopathi- or nucleotides and/or viral nucleic acids generally have city, virus yield and plaque formation of live and/or broad-spectrum activity against a wide range of CoVs pseudotyped CoVs. Examples of drugs identified using and other viruses (TABLE 2). Mycophenolate mofetil is this approach include interferon alfa, interferon beta, an anti-rejection drug that inhibits inosine monophos- interferon gamma, ribavirin and inhibitors of cyclo phate dehydrogenase and the synthesis of guanine philin8,74,116–122. These drugs have the obvious advantage of monophosphate122. The active compound, mycophenolic being readily available with known pharmacokinetic and acid, exhibits antiviral activity in vitro against various pharmacodynamic properties, side effects and dosing viruses, including hepatitis B virus (HBV), hepatitis C regimens. However, they do not have specific anti-CoV virus (HCV) and arboviruses122. Mycophenolic acid was effects and may be associated with severe adverse effects. identified as a potential anti-MERS-CoV drug using The second anti-CoV drug discovery approach high-throughput screening and has potent anti-MERS- involves the screening of chemical libraries comprising CoV activity in vitro122. However, a subsequent study in large numbers of existing compounds or databases that a non-human primate model showed that MERS-CoV- contain information on transcriptional signatures in infected common marmosets treated with mycopheno different cell lines122–127. This approach provides rapid, late mofetil had a worse outcome with more severe high-throughput screening of many readily available disease and higher viral loads in necropsied lung and compounds that can then be further evaluated by anti- extrapulmonary tissues than untreated animals did128. viral assays. Various classes of drugs have been identified Renal transplant recipients who were on maintenance in these drug repurposing programmes, including many mycophenolate mofetil therapy also developed severe that have important physiological and/or immunological or fatal MERS129,130. Thus, the usual dosage of myco effects such as those that affect neurotransmitter regu- phenolate mofetil monotherapy is unlikely to be useful lation, the oestrogen receptor, kinase signalling, lipid or for prophylaxis or treatment of CoV infections.

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mAbs targeting S1 mAbs, peptides and TMPRSS2 inhibitors or S2 (REGN3051) Attachment small molecules (camostat mesylate) and entry against host receptor Cell surface S2 subunit antiviral to inhibit entry non-endosomal peptides (HR2P) Host (NAAE, YS110) pathway receptor Furin inhibitors (dec-RVKR-CMK)

Clathrin-mediated endocytosis inhibitors (chlorpromazine, ouabain) Endosomal Endosomal pathway acidiﬁcation Cathepsin inhibitors (E64D) inhibitors (chloroquine)

Uncoating

PLpro Genomic RNA 5ʹ C 3ʹ (+) 3CLpro

Translation of ORF1a/b PLpro inhibitors (GRL0617) Helicase inhibitors (SSYA10-001) 3CLpro inhibitors (lopinavir) RdRp inhibitors (ribavirin, BCX4430)

pp1a Replication– pp1ab nsp1–16 transcription complex CYP–calcineurin– CYP inhibitors (alisporivir) NFAT pathway Membrane-bound RNA synthesis inhibitors (K22) Viral nucleic acid synthesis siRNAs against structural and inhibitors (MPA, DRACO) mRNA synthesis accessory genes (SARS-CoV S, and splicing E, M, N, ORF3a/7a/7b)* Genome replication Transcription 3ʹ 5ʹ (–) and replication 3ʹ 5ʹ (–)

5ʹ 3ʹ (+) 5ʹ C 3ʹ (+)

Translation

AP S E M N N Budding and assembly Rough ER

ERGIC Nucleus

ERK–MAPK and PI3K–AKT–mTOR signalling inhibitors (trametinib) Golgi Vesicle

Exocytosis

Membrane-binding photosensitizer (LJ001) Recombinant Host-based treatment options Interferon inducers interferon alfa Virus-based treatment options (poly (I:C), nitazoxanide) and interferon beta

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◀ Figure 2 | Virus-based and host-based treatment options targeting the coronavirus integral component of the CoV replication complex that replication cycle. Binding between the receptor-binding domain on the S1 subunit of is involved in DMV formation, including nsp6H121L and spike glycoprotein (S) and the host receptor triggers conformational changes in the S2 nsp6M159V (REF. 112). The emergence of K22 resistance subunit of S. This leads to fusion of the viral and cell membranes. Coronaviruses (CoVs) should be monitored in subsequent in vivo studies. enter the host cell using the endosomal pathway and/or the cell surface non-endosomal Recently, a new class of broad-spectrum antivirals that pathway. Endosomal cell entry of CoVs is facilitated by low pH and the pH-dependent endosomal cysteine protease cathepsins. S is activated and cleaved into the S1 and S2 targets long viral double-stranded RNA (dsRNA) has subunits by other host proteases, such as transmembrane protease serine 2 (TMPRSS2) been reported. For example, dsRNA-activated caspase and TMPRSS11D, which enables cell surface non-endosomal virus entry at the plasma oligomerizer (DRACO) is a chimeric protein with a viral membrane. Middle East respiratory syndrome (MERS)-CoV S is additionally activated dsRNA-binding domain and a pro-apoptotic domain that by the serine endoprotease furin. CoVs then dissemble intracellularly to release the selectively induces apoptosis in cells that contain viral nucleocapsid and viral RNA into the cytoplasm for the translation of ORF1a/b into dsRNA but spares uninfected host cells132. DRACO is the large replicase polyprotein 1a (pp1a) and pp1ab and for the replication of genomic active against many RNA viruses in vitro and/or in vivo132. RNA. pp1a and pp1ab are cleaved by papain-like protease (PLpro) and 3C‑like protease If an effective mode of DRACO delivery can be achieved, (3CLpro) to produce non-structural proteins (NSPs), including RNA-dependent RNA a broad-spectrum anti-CoV drug that targets highly polymerase (RdRp) and helicase, which are involved in the transcription and replication conserved CoV RNA sequences might become a reality. of the virus. The NSPs produced by the cleavage of pp1a and pp1ab form the replication–transcription complex. Attachment of the hydrophobic domains of the CoV replication–transcription complex to the limiting membrane derived from Viral enzymes. All of the major enzymes and proteins of the endoplasmic reticulum (ER) produces typical CoV replication structures including CoVs that are involved in viral replication are potentially double-membrane vesicles and convoluted membranes. The full-length positive-strand druggable targets (TABLE 2). The SARS-CoV and MERS- genomic RNA is transcribed to form a full-length negative-strand template for synthesis CoV PLpro enzymes exhibit proteolytic, deubiquitylat- of new genomic RNAs and overlapping subgenomic negative-strand templates. ing and deISGylating activities133–135. Crystallography has Subgenomic mRNAs are then synthesized and translated to produce the structural and facilitated the characterization of these PLpro enzymes accessory proteins. The helical nucleocapsid formed by the assembly of nucleocapsid and the identification of PLpro inhibitors136. Numerous protein (N) and genomic RNA interacts with the other structural proteins to form the SARS-CoV PLpro inhibitors belonging to different classes assembled virion, which is then released by exocytosis into the extracellular have been identified, including small-molecule inhibitors, compartment. Virus- and host-based treatment options are highlighted in red and blue, respectively. +, positive-strand RNA; -, negative-strand RNA; AP, accessory protein; thiopurine compounds, natural products, zinc ion and 137 CYP, cyclophilin; dec-RVKR-CMK, decanoyl-Arg-Val-Lys-Arg-chloromethylketone; zinc conjugate inhibitors and naphthalene inhibitors . DRACO, double-stranded RNA-activated caspase oligomerizer; E, envelope protein; However, some of these drugs only inhibit the enzymatic ER, endoplasmic reticulum; ERGIC, endoplasmic reticulum Golgi intermediate activities of PLpro without inhibiting viral replication, compartment; ERK, extracellular signal-regulated kinase; M, membrane; mAb, or vice versa137–139. None has been validated in animal or monoclonal antibody; MAPK, mitogen-activated protein kinase; MPA, mycophenolic human studies137,138. Furthermore, most PLpro inhibitors acid; mTOR, mammalian target of rapamycin; N, nucleocapsid protein; NAAE, have narrow-spectrum activity because of the struc- N-(2‑aminoethyl)-1‑aziridine-ethanamine; NFAT, nuclear factor of activated T cells; tural differences among the PLpro enzymes of different ORF, open reading frame; PI3K, phosphoinositide 3‑kinase; poly(I:C), polyinosinic:poly‑ CoVs140,141. For example, most SARS-CoV PLpro inhibi- cytidylic acid; RdRp, RNA-dependent RNA polymerase; S, spike glycoprotein; tors are inactive against MERS-CoV because of the struc- SARS-CoV, severe acute respiratory syndrome coronavirus; siRNA, small interfering RNA. *Only siRNAs that have been evaluated in published reports are included. siRNAs turally different, flexible blocking loop 2 (BL2) domains 140 directed against other parts of the CoV genome would also be expected to diminish in the PLpro enzymes of SARS-CoV and MERS-CoV . the accumulation or translation of genomic and all upstream subgenomic RNAs. 3CLpro is the other major CoV protease that cleaves Adapted with permission from REF. 9, American Society for Microbiology. the large replicase polyproteins during viral replication. SARS-CoV 3CLpro can be targeted by numerous classes of protease inhibitors, including zinc or Ribozymes (also known as catalytic RNA or RNA mercury conjugates, C2‑symmetric diols, peptidomi- enzymes) are RNA molecules that catalyse specific bio- metic‑α,β‑unsaturated esters, anilides, benzotriazole, chemical reactions. A chimeric DNA–RNA hammerhead N‑phenyl‑2‑acetamide, biphenyl sulfone, glutamic acid ribozyme that specifically recognizes the base sequence and glutamine peptides with a trifluoromethylketone GUC, which is present in the loop region of SARS-CoV group, pyrimidinone and pyrazole analogues142. Some mRNA, substantially reduces the expression of recom- of these 3CLpro inhibitors demonstrate broad-spectrum binant SARS-CoV RNA in vitro131. However, ribozymes in vitro activities against CoVs with highly similar are rapidly degraded in vivo and delivery methods would key residues for substrate recognition at their 3CLpro have to be optimized in humans before ribozymes could enzymes143,144. Among these 3CLpro inhibitors, the most become clinically useful. readily available one is lopinavir, a protease inhibitor Agents targeting the specific host cell membrane- used to treat HIV infections that is usually marketed as a bound CoV replication complex have also been ritonavir-boosted form (lopinavir–ritonavir). Lopinavir investigated. One such compound, K22, inhibits and/or lopinavir–ritonavir have anti-CoV activity in vitro, membrane-bound CoV RNA synthesis and is active as well as in MERS-CoV-infected non-human primates against a broad range of CoVs in vitro112. In cell culture, and in non-randomized trials of SARS patients76,77,123,128,145. K22 exerts potent anti-CoV activity during an early step It is postulated that the 3CLpro-inhibiting activity of of the viral replication cycle and impairs formation of lopinavir–ritonavir contributes at least partially to its anti- DMVs112. HCoV‑229E escape mutants that are resistant CoV effects146. It remains to be seen if resistance emerges, to K22 have substitutions in the potential membrane- as it has in patients with HIV infection, when lopinavir– spanning domains in nsp6, a membrane-spanning ritonavir is routinely used to treat CoV infections.

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Table 2 | Representative virus-based treatment strategies for CoV infections Targeted viral Examples Mechanism of action Status Comments Refs components Viral nucleic acids Nucleosides Mycophenolic acid Inhibitor of IMPDH and guanine Marketed • Broad spectrum: MERS-CoV, HBV, HCV, 122,128, and/or monophosphate synthesis arboviruses (JEV, WNV, YFV, dengue virus 192 nucleotides and CHIKV) • Worsened outcome in MERS-CoV-infected common marmosets • Unlikely to be useful as monotherapy, but combinatorial therapy with interferon beta‑1b is synergistic in vitro mRNA Ribozyme An antisense RNA with catalytic Preclinical • Narrow spectrum 131 activity that specifically • Optimal delivery method in humans recognizes the base sequence is uncertain GUC in the loop region on the mRNA of CoVs Host cell K22 Inhibitor of membrane-bound Preclinical • Broad spectrum: SARS-CoV, MERS-CoV, 112 membrane-bound RNA synthesis and double HCoV‑229E and animal CoVs viral replication membrane vesicle formation • No animal or human data available complex Long viral dsRNA DRACO A chimeric protein with a viral Preclinical • Broad spectrum: adenoviruses, arenaviruses, 132 dsRNA-binding domain and bunyaviruses, dengue virus, IAV, a pro-apoptotic domain that picornaviruses, rhinoviruses and reoviruses selectively induces apoptosis in • Anti-CoV activity has yet to be cells containing viral dsRNA demonstrated Viral enzymes PLpro GRL0617, Inhibitors of PLpro activity Preclinical • Narrow spectrum 137–140 compound 4 • No animal or human data available 3CLpro Lopinavir, N3, CE‑5 Inhibitors of 3CLpro activity Preclinical • Broad spectrum: SARS-CoV, MERS-CoV, 76,77, and GRL‑001 HCoV‑229E, HCoV‑NL63 and animal CoVs 123,128, • Marketed: lopinavir–ritonavir 143–146, • Improved outcome of MERS-CoV-infected 275,276 common marmosets • Improved outcome of SARS patients in non-randomized trials RdRp Ribavirin Guanosine analogue that Marketed • Broad spectrum: many viral infections, 10,21, inhibits viral RNA synthesis especially SARS, MERS, RSV, HCV and viral 86–89, and mRNA capping haemorrhagic fevers 117, • Active against SARS-CoV and MERS-CoV 277–280 at high doses in vitro • Benefits in SARS and MERS patients are uncertain • Side effects are common and may be severe with high-dose reigmens BCX4430 Adenosine analogue that acts Preclinical • Broad spectrum: SARS-CoV, MERS-CoV, 149 as a non-obligate RNA chain IAV, filoviruses, togaviruses, bunyaviruses, terminator to inhibit viral RNA arenaviruses, paramyxoviruses, polymerase function picornaviruses and flaviviruses • No animal or human data are available for CoVs Fleximer nucleoside Doubly flexible nucleoside Preclinical • Active against MERS-CoV and HCoV‑NL63 150 analogues of analogues based on the acyclic • Further modification of existing nucleoside acyclovir sugar scaffold of acyclovir analogues with different fleximers is and the flex-base moiety in possible fleximers that inhibit RdRp • No animal or human data available siRNA* Short chains of dsRNA that Preclinical • Narrow spectrum 151,152 interfere with the expression • Optimal delivery method in humans is of RdRp uncertain Helicase Bananins and Inhibits helicase unwinding and Preclinical • Possibly broad spectrum: helicase is 153,154 5‑hydroxychromone ATPase activities relatively conserved among CoVs derivatives • High risk of toxicity SSYA10‑001 and Inhibits helicase unwinding Preclinical • Broad spectrum: SARS-CoV, MERS-CoV 155,156, ADKs without affecting ATPase and animal CoVs 281 activity • Likely to be less toxic than bananins and 5‑hydroxychromone derivatives

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Table 2 (cont.) | Representative virus-based treatment strategies for CoV infections Targeted viral Examples Mechanism of action Status Comments Refs components Viral spike glycoprotein RBD of the S1 MERS‑4, MERS‑27, mAbs against the RBD of the Preclinical • Narrow spectrum 94–97, subunit of S m336, m337, m338, S1 subunit that block virus–host • May reduce the need for 100, REGN3051 and cell binding convalescent-phase plasma therapy 160–162 REGN3048 mAbs • Protective effects demonstrated in animal models S2 subunit of S HR2P and P1 Antiviral peptides that Preclinical • Narrow spectrum 92,93, peptides inhibit fusion of S with host • Enfuvirtide, an anti-HIV antiviral peptide 99, cell receptor fusion inhibitor, has been successfully 160–162 marketed Oligosaccharides Griffithsin A carbohydrate-binding agent Preclinical • Broad spectrum: SARS-CoV, MERS-CoV, 173,174 on S that specifically binds to HCoV‑229E, HCoV‑OC43, HIV, HCV oligosaccharides on S, thereby and Ebola virus blocking virus–host cell binding • Well tolerated in rodents S expression siRNA* Short chains of dsRNA that Preclinical • Narrow spectrum 169–172 interfere with the expression • SARS-CoV-infected rhesus macaques had of SARS-CoV S better clinical, virological, and histological parameters • Optimal delivery method in humans is uncertain Viral envelope, membrane, nucleocapsid and accessory proteins E siRNA* Short chains of dsRNA that Preclinical • Narrow spectrum 179 interfere with the expression • Optimal delivery method in humans is of SARS-CoV E uncertain Hexamethylene Viroporin inhibitor that Preclinical • Inhibited ion channel activities of SARS-CoV, 181,182 amiloride inhibits the ion channel HCoV‑229E and some animal CoVs activity of CoV E • Analogue of the potassium-sparing diuretic drug amiloride M siRNA* Short chains of dsRNA that Preclinical • Narrow spectrum 179 interfere with the expression • Optimal delivery method in humans is of SARS-CoV M uncertain N PJ34, intrabodies‡ Reduces the RNA-binding Preclinical • Narrow spectrum 179,183, and siRNA* affinity of N and viral replication • Optimal delivery method in humans is 282 uncertain Accessory siRNA* Short chains of dsRNA that Preclinical • Narrow spectrum 180 proteins interfere with the expression • Optimal delivery method in humans is of proteins from SARS-CoV uncertain ORF3a, ORF7a and ORF7b Lipid membrane LJ001 and JL103 Membrane-binding Preclinical • Broad spectrum: enveloped viruses 184–187 photosensitizers that induce (IAV, filoviruses, poxviruses, arenaviruses, singlet oxygen modifications bunyaviruses, paramyxoviruses, of specific phospholipids flaviviruses and HIV‑1) • Anti-CoV activity has yet to be demonstrated 3CLpro, 3C‑like protease; ADK, aryl diketoacid; CHIKV, Chikungunya virus; CoV, coronavirus; DRACO, double-stranded RNA activated caspase oligomerizer; dsRNA, double-stranded RNA; E, envelope protein; HBV, hepatitis B virus; HCoV, human coronavirus; HCV, hepatitis C virus; IAV, influenza A virus; IMPDH, inosine-monophosphate dehydrogenase; JEV, Japanese encephalitis virus; M, membrane protein; mAb, monoclonal antibody; MERS, Middle East respiratory syndrome; N, nucleocapsid protein; ORF, open reading frame; PLpro, papain-like protease; RBD, receptor-binding domain; RdRp, RNA-dependent RNA polymerase; RSV, respiratory syncytial virus; S, spike glycoprotein; SARS, severe acute respiratory syndrome; siRNA, small interfering RNA; WNV, West Nile virus; YFV, yellow fever virus. *Only siRNAs that have been evaluated in published reports are included. siRNAs directed against other parts of the CoV genome would also be expected to diminish the accumulation or translation of genomic and upstream subgenomic RNAs. ‡Intrabodies are antibodies that work within the cell to bind to intracellular proteins.

RdRp is an essential part of the CoV replication– of mutations in RNA-dependent viral replication are transcription complex and is involved in the production considered to be important for RNA viruses, includ- of genomic and subgenomic RNAs. Ribavirin is a guano- ing CoVs147. High-dose ribavirin has been used to treat Convalescent-phase plasma sine analogue with broad-spectrum antiviral activity SARS patients, but the benefits were unclear8,10,21,72,74,75,117. Plasma that contains and has been used in the treatment of severe respiratory It exhibits moderate anti-MERS-CoV activity at high neutralizing antibodies against a microorganism and is syncytial virus infection, HCV infection and viral haem- doses in vitro and in MERS-CoV-infected rhesus obtained from patients orrhagic fevers. Its exact mechanism of action is undeter- macaques, but there was no obvious survival benefit in recovering from the infection. mined, but inhibition of mRNA capping and induction small cohorts of MERS patients86–89,121,148. Moreover, the

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severe side effects associated with the use of high-dose results from treatment with products containing low ribavirin limit its clinical application in patients with antibody titres has been reported in cell line and animal severe CoV infections8,74. Recently, a novel adenosine studies158,159. To overcome these problems, mAbs target- analogue, BCX4430 (Immucillin‑A), was developed149. ing different regions of SARS-CoV S have been generated It acts as a non-obligate RNA chain terminator to inhibit by immunization of human immunoglobulin transgenic viral RNA polymerases of a wide range of RNA viruses, mice, cloning of small chain variable regions from naive including CoVs such as SARS-CoV and MERS-CoV as and convalescent patients as well as from immortaliza- well as filoviruses such as Ebola and Marburg viruses149. tion of convalescent S‑specific B cells160. Most of these Its development for human use has been fast-tracked to mAbs target specific epitopes on the S1 subunit RBD increase the number of treatment options for the recent to inhibit virus–cell receptor binding, whereas others Ebola virus epidemic in West Africa. Existing nucleoside bind to the S2 subunit to interrupt virus–cell fusion160. analogues, such as acyclovir, could be modified by incor- Regardless of their binding sites and mechanisms, these porating fleximers, which have increased binding affinity mAbs exhibit neutralizing activities and reduced viral and can overcome resistance caused by point mutations titres in vitro and/or in small animal models. Similarly, in biologically important binding sites150. These acyclic several mAbs targeting different epitopes on the S1 sub- fleximer nucleoside analogues inhibit MERS-CoV and unit RBD of MERS-CoV S have been developed94–97,100. HCoV‑NL63 in vitro at micromolar concentrations150. These monoclonal antibodies bind to the RBD with Notably, resistance to nucleoside analogues due to muta- 10‑fold to >450‑fold higher affinity than does human tions in RdRp has been reported for other RNA viruses, DPP4, resulting in broader and higher neutralizing activ- and should be monitored when these agents are used to ity in vitro. Importantly, combination therapy with two or treat CoV infections. In addition to nucleoside analogues, more synergistically acting humanized or human mAbs siRNA molecules targeting SARS-CoV RdRp have been targeting non-cross-resistant epitopes or different regions used to inhibit SARS-CoV in vitro151,152. of S may help to reduce the frequency with which viruses Helicase catalyses the unwinding of duplex oligo mutate to escape antibody-mediated neutralization94. nucleotides into single strands in an ATP-dependent Treatment with these mAbs showed protective effects reaction during the CoV replication cycle. Helicase inhib- in MERS-CoV-infected human DPP4-transgenic mice itors are attractive anti-CoV treatment options because and mice transduced by adenoviral vectors expressing the helicases of different CoVs are highly homologous. human DPP4 (REFS 100,161,162). Their safety profiles and Based on their mechanisms of action, CoV helicase inhib- treatment effects in patients should be further evaluated. itors can be broadly categorized into two groups. The Antiviral peptides targeting different regions of S are first group includes bananins and 5‑hydroxychromone another promising therapeutic strategy. The S2 subunits derivatives, which inhibit the unwinding and ATPase or stalk regions of both SARS-CoV and MERS-CoV activity of SARS-CoV helicase, resulting in inhibition S are class I viral fusion proteins that each contain an of viral replication in vitro153,154. However, the toxic- N‑terminal fusion peptide, heptad repeat 1 (HR1) and ity resulting from the inhibition of cellular ATPases or HR2 domains, a transmembrane domain and a cyto- kinases by these compounds has limited their develop- plasmic domain92. Antiviral peptides analogous to the ment for human use. The second group of CoV helicase N terminus, pre-transmembrane domain or the loop inhibitors includes compounds that selectively inhibit region separating the HR1 and HR2 domains of SARS- the unwinding activity but not the ATPase activity of CoV inhibited virus plaque formation by 40–80% at CoV helicase. An example is SSYA10‑001, a triazole that micromolar concentrations163,164. Similarly, antiviral pep- inhibits a broad range of CoVs, including SARS-CoV, tides spanning the HR2 domain of MERS-CoV inhibit MERS-CoV and mouse hepatitis virus155,156. The toxicity S-mediated cell–cell fusion and viral entry into cells of SSYA10‑001 should be evaluated in animal models. in vitro92,93. A peptide called HP2P‑M2 that is derived from the HR2 domain, if administered intranasally before Viral spike glycoprotein. The membrane-anchored glyco- or after viral challenge, protected C57BL/6 mice and mice protein, S, is a major immunogenic antigen and is essen- deficient for V(D)J recombination-activating protein 1 Viroporin tial for the interaction between the virus and the host (RAG1) that were recently transduced by adenoviral A small integral membrane cell receptor (FIG. 2). Adoptive transfer of sera containing vectors expressing human DPP4 from MERS-CoV infec- protein that is localized anti-MERS-CoV-S antibodies blocked virus attachment tion with 10‑fold to >1,000‑fold reduction in viral titres primarily within the endoplasmic reticulum and and accelerated viral clearance from the lungs of MERS- in the lung; this protection was enhanced by combining 99 plasma membranes of host CoV infected BALB/c mice that were recently transduced this peptide with interferon beta . Combining antiviral cells, and has the characteristic by adenoviral vectors expressing human DPP4 (REF. 157) peptides targeting different regions of the S2 subunit may ability to form ion channels (TABLE 2). Small cohorts of SARS patients who received be synergistic in vitro and overcome the theoretical risk or pores. convalescent-phase plasma containing neutralizing anti- of drug resistance165. Importantly, an analogous fusion Protein cage nanoparticles bodies that probably targeted CoV S had significantly inhibitor, enfuvirtide, which binds to glycoprotein 41 of Nanoscale delivery platforms, higher discharge rates by 3 weeks after symptom onset HIV to block membrane fusion and HIV cell entry, has made of biomaterials and/or and a lower mortality rate83,84. However, the use of conva- been successfully marketed for treatment of HIV‑1 infec- proteins, that are used for lescent-phase plasma therapy during emerging CoV out- tion166. Primary resistance to enfuvirtide is rare and can various biomedical applications including the breaks is limited by the good will of convalescent patients be overcome by modifying the drug such that it contains 167,168 delivery of therapeutic with high serum neutralizing antibody titres. Disease secondary compensatory mutations . This example cargo molecules. worsening associated with immune enhancement that of successful drug development includes measures to

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counteract drug resistance and therefore favours antiviral SARS-CoV and MERS-CoV in vitro and in animal mod- peptides over anti-CoV S siRNAs for further evaluation els8,99,116,121,122,128,148,191,192. Various combinations of inter- in vivo; siRNAs have remained in preclinical develop- feron alfa or interferon beta with other antivirals such as ment despite their reported antiviral activities in vitro and ribavirin and/or lopinavir–ritonavir have been used to in SARS-CoV-infected rhesus macaques owing to the treat patients with SARS or MERS. Overall, combination lack of reliable drug delivery methods in humans169–172. treatments consisting of interferons and ribavirin did not Another class of anti-CoV agents that target S to consistently improve outcomes8,9,74,86,87,89. The apparent inhibit CoV entry is the carbohydrate-binding agents. discrepancy between in vitro findings and in vivo out-

Griffithsin is an antiviral protein originally isolated from comes may be related to the high EC50/Cmax ratios of these the red alga Griffithsia spp.173. It binds specifically to drugs and the delay between symptom onset and drug oligosaccharides on viral surface glycoproteins such as administration8,121,122. This delay is especially relevant S and HIV glycoprotein 120. It inhibits a broad range of for MERS patients, as they have a much shorter median CoVs, including SARS-CoV, HCoV‑229E, HCoV‑OC43 time interval between symptom onset and death than and HCoV‑NL63 in vitro and in SARS-CoV-infected do SARS patients9,58. The use of recombinant interferon 173,174 mice . The optimal delivery modes and safety profiles beta‑1b, which has the lowest EC50/Cmax ratio against of these agents in humans should be further evaluated. MERS-CoV among tested preparations of recombinant interferons, should be evaluated in combination with Viral envelope, membrane, nucleocapsid and acces- other effective antivirals in clinical trials at early stages of sory proteins. E, M and N and some of the accessory the infection122,128. proteins are not only essential for virion assembly but Polyinosinic:polycytidylic acid (poly(I:C)) is a syn- may also have additional functions that suppress the thetic analogue of dsRNA that strongly induces type I host immune response to facilitate viral replication. For interferons. It substantially reduced the MERS-CoV load example, the accessory proteins 4a and 4b, and possibly in BALB/c mice that were transduced by adenoviral vec- also M and accessory protein 5 of MERS-CoV, exhibit tors expressing human DPP4 shortly before poly(I:C) interferon antagonist activities, and SARS-CoV N acts as administration, although its effects in standard cell cul- a viral suppressor of RNA silencing and suppresses RNA ture protection assays are not published157. Intramuscular interference triggered by either short hairpin RNAs or injection of poly(I:C) stabilized with poly‑L‑lysine and siRNAs175–178 (TABLE 2). siRNAs targeting E, M, N, ORF3a, carboxymethylcellulose seemed to be well tolerated by ORF7a or ORF7b of SARS-CoV inhibited viral replica- patients with malignant gliomas in Phase II clinical tion in vitro179,180. However, similar to anti-CoV S siR- trials193,194. Nitazoxanide is another potent type I inter- NAs, none of these siRNAs is ready for human use until feron inducer that has been used in humans for parasitic better delivery methods become available. infections195. It is a synthetic nitrothiazolyl–salicylamide Alternatively, an increasing number of agents that derivative that exhibits broad-spectrum antiviral activ- target specific binding sites or functions of these proteins ities against both RNA and DNA viruses including are being generated through crystallography and func- canine CoV, influenza viruses, HBV, HCV, HIV, rota tional assays. Examples include the viroporin inhibitor virus, norovirus and flaviviruses195. It has been evaluated hexamethylene amiloride, which reduces the ion chan- in Phase II and Phase III clinical trials for the treatment nel activity of E in SARS-CoV and HCoV‑229E, and of HCV infection and influenza and has a good safety PJ34, which binds to a distinct ribonucleotide-binding profile195–197. Other innate immunomodulators that have pocket at the N‑terminal domain of N in HCoV‑OC43 anti-SARS-CoV effects in animal models include the (REFS 181–183). However, these agents are likely to be antimicrobial peptide rhesus θ‑defensin 1 and protein narrow-spectrum as the binding sites and functions of cage nanoparticles that elicit a host immune response these proteins are unique to individual CoVs. in inducible bronchus-associated lymphoid tissue198,199. Novel lipophilic thiazolidine derivatives, such as LJ001 The combined use of interferon inducers and innate and JL103, are membrane-binding photosensitizers that immunomodulators with effective antiviral agents may produce singlet oxygen molecules to induce changes in be synergistic and should be evaluated in animal models. the properties of lipid membranes and prevent fusion between viral and target cell membranes. They exhibit Other host signalling pathways involved in viral repli- broad-spectrum activities against numerous enveloped cation. In addition to direct potentiation of the inter- viruses and may be active against CoVs184–187. feron response, other cell signalling pathways have been identified as potential anti-CoV treatment targets Host-based anti-CoV treatment options (TABLE 3). Cyclophilins interact with SARS-CoV nsp1 to Broad-spectrum host innate immune response. The host modulate the calcineurin pathway, which is important innate interferon response is crucial for the control of in the T cell-mediated adaptive immune response120. viral replication after infection188. Although CoVs are The calcineurin inhibitor cyclosporine inhibits a broad able to suppress the interferon response for immune range of CoVs in vitro118–120. However, its clinical appli- evasion, they remain susceptible to interferon treatment cation is limited by its immunosuppressive effects and 189,190 in vitro . The interferon response can be augmented high EC50/Cmax ratio at standard therapeutic dosages. The by the administration of recombinant interferons or antiviral activities of newer, non-immunosuppressive cal- interferon inducers (TABLE 3). Recombinant interferon cineurin inhibitors, which are active against HCoV‑NL63, alfa and interferon beta inhibit the replication of both should be evaluated for SARS-CoV and MERS-CoV200.

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Table 3 | Representative host-based treatment strategies for CoV infections Targeted host Examples Mechanism of action Status Comments Refs factors Broad-spectrum host innate immune response Interferon Recombinant Exogenous interferons Marketed • Broad spectrum against many CoVs 8,9,74,86,87, response interferons and other viruses 89,99,116, (interferon alfa, • Recombinant interferon beta was more 121,122,128, interferon beta, potent than interferon alfa for SARS-CoV 148,191,192, interferon gamma) and MERS-CoV in vitro 215 • Interferon alfa reduced viral titres in lungs of SARS-CoV-infected mice and cynomolgus macaques • Intranasal interferon beta administered before or after MERS-CoV challenge reduced viral titres in the lungs of Ad5‑hDPP4 C57BL/6 and Rag1–/– mice by 10–100 fold • Subcutaneous interferon beta‑1b improved outcomes of MERS-CoV-infected common marmosets • Benefits for SARS patients are uncertain • Benefits of interferon alfa‑2a, interferon alfa‑2b and interferon beta‑1a for MERS patients are uncertain Poly(I:C) Induces interferon Phase II clinical • Reduced MERS-CoV load in Ad5‑hDPP4 157,193,194 production trials BALB/c mice • Used in Phase II clinical trials of patients with malignant gliomas Nitazoxanide A thiazolide that Marketed • Broad spectrum: canine CoV, IAV, IBV, RSV, 195 induces the host innate PIF, Sendai virus, rhinovirus, norovirus, immune response rotavirus, Dengue virus, JEV, YFV, HBV, by potentiation HCV and HIV of interferon alfa • Used in patients with parasitic infections and interferon and in Phase II and III clinical trials of HCV beta production infection and influenza by fibroblasts and • Activity against human-pathogenic CoVs activation of PKR has yet to be determined Other host signalling pathways involved in viral replication Cyclophilins Cyclosporine, Cyclophilin inhibitor Marketed • Broad spectrum: CoVs (SARS-CoV, 118‑120,200 alisporivir that could modulate MERS-CoV, HCoV‑NL63, HCoV‑229E, the interaction of and animal CoVs), HIV, HCV, HPV, vaccinia cyclophilins with virus and VSV SARS-CoV nsp1 and • Alisporivir does not have the the calcineurin–NFAT immunosuppressive effects of pathway cyclosporine and may therefore be a more suitable antiviral candidate Kinase signalling Trametinib, Kinase signalling Marketed • Active against SARS-CoV and MERS-CoV 124,125 pathways selumetinib, inhibitors that block • May be associated with everolimus, the ABL1, ERK–MAPK immunopathology rapamycin, and/or PI3K–AKT– dasatinib and mTOR pathways, imatinib which may block early viral entry and/or post-entry events Host receptors utilized by CoVs for viral entry ACE2 P4 and P5 peptides ACE2‑derived peptides Marketed • Narrow spectrum: SARS-CoV 202,203 and NAAE or small molecules • May affect important biological functions targeting ACE2 that such as blood pressure regulation block SARS-CoV S-mediated cell fusion DPP4 Anti‑DPP4 mAb Anti‑DPP4 mAbs that Phase I clinical • Narrow spectrum: MERS-CoV 102,201,227 clones 2F9 and block MERS-CoV trial • May affect important biological YS110 S-mediated cell fusion functions such as glucose metabolism and immunological responses • mAb clone YS110 was used in a Phase I clinical trial of patients with advanced malignancies

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Table 3 (cont.) | Representative host-based treatment strategies for CoV infections Targeted host Examples Mechanism of action Status Comments Refs factors Host proteases utilized by CoVs for viral entry Endosomal E64D, K11777 and Cathepsin inhibitors Preclinical • Broad spectrum: CoVs (SARS-CoV and 111,124,127, protease (for the small molecule that block endosomal MERS-CoV), filoviruses (Ebola virus) and 283 example, 5705213 protease-mediated paramyxoviruses (Hendra and Nipah cathepsins) cleavage and viruses) the endosomal • Combination with TMPRSS2 inhibitors entry pathway necessary for complete inhibition of MERS-CoV in vitro Surface protease Camostat mesylate TMPRSS2 inhibitor Marketed • Broad spectrum: CoVs (SARS-CoV, 109,111,207, (for example, that blocks the MERS-CoV and HCoV‑229E), IAV and PIF 208,284–286 TMPRSS2) TMPRSS2‑mediated cell • Combination with cathepsin inhibitors surface entry pathway is necessary for complete inhibition of MERS-CoV in vitro • Used to treat patients with chronic pancreatitis Other host dec-RVKR-CMK Furin inhibitor that Preclinical • Active against MERS-CoV and may be 110 proteases (for blocks furin-mediated active against other CoVs that utilize furin example, furin) cleavage of S for S cleavage Endocytosis Clathrin-mediated Chlorpromazine An antipsychotic that Marketed • Broad spectrum: SARS-CoV, MERS-CoV, 123 endocytosis also affects the assembly HCV and alphaviruses of clathrin-coated pits at • Clinical benefit uncertain owing to a high

the plasma membrane EC50/Cmax ratio at the usual therapeutic dosages Clathrin-mediated Ouabain and ATP1A1‑binding Marketed • Active against MERS-CoV at nanomolar 209 endocytosis bufalin cardiotonic concentrations in vitro steroids that inhibit • May have risk of toxicity clathrin-mediated endocytosis Endosomal Chloroquine An antimalarial that Marketed • Broad spectrum: CoVs (SARS-CoV, 123, acidification sequesters protons in MERS-CoV, HCoV‑229E and 210–215 lysosomes to increase HCoV‑OC43), HIV, flaviviruses and Ebola, the intracellular pH Hendra and Nipah viruses in vitro • Not active against SARS-CoV-infected mice

ACE2, angiotensin-converting enzyme 2; Ad5‑hDPP4, adenovirus type 5 expressing human dipeptidyl peptidase 4; ATP1A1, ATPase subunit α1; Cmax, peak serum concentration; CoV, coronavirus; dec-RVKR-CMK, decanoyl-Arg-Val-Lys-Arg-chloromethylketone; DPP4; dipeptidyl peptidase 4; EC50, half-maximal effective concentration; ERK, extracellular signal-regulated kinase; HBV, hepatitis B virus; HCoV, human coronavirus; HCV, hepatitis C virus; HPV, human papillomavirus; IAV, influenza A virus; IBV, influenza B virus; JEV, Japanese encephalitis virus; mAb, monoclonal antibody; MAPK, mitogen-activated protein kinase; MERS, Middle East respiratory syndrome; mTOR, mammalian target of rapamycin; NAAE, N-(2‑aminoethyl)-1‑aziridine-ethanamine; NFAT, nuclear factor of activated T cells; nsp1, non-structural protein 1; PI3K, phosphoinositide 3‑kinase; PIF, parainfluenza virus; PKR, protein kinase R; poly(I:C), polyinosinic:polycytidylic acid; RSV, respiratory syncytial virus; S, spike glycoprotein; SARS, severe acute respiratory syndrome; TMPRSS2, transmembrane protease serine 2; VSV, vesicular stomatitis virus; YFV, yellow fever virus.

Similarly, agents that modulate other cellular signalling ACE2 and SARS-CoV S-mediated cell–cell fusion pathways, such as kinase signalling pathway inhibitors, in vitro202. Synthetic peptides analogous to critical seg- also exhibit anti-CoV activities and are commercially ments of ACE2 also have anti-SARS-CoV activity at available124,125. However, their toxicities may limit their micromolar concentrations in vitro203. However, none of use in patients with severe CoV infections. these receptor-directed compounds has yet been tested in patients with CoV infections. Their anti-CoV activity Host factors utilized by CoVs for viral replication. CoVs is likely to be narrow-spectrum, as different CoVs util utilize specific host factors for virus entry and replication ize different host cell receptors. Furthermore, the risks (FIG. 2). The host receptor can be targeted by specific mon- of immunopathology must be assessed, especially given oclonal or polyclonal antibodies, peptides or functional the multiple essential biological and immunological inhibitors (TABLE 3). For example, anti‑DPP4 mAbs inhibit functions of these receptors. MERS-CoV cell entry in vitro201. YS110 is a recombinant The entry of CoVs into host cells via the endosomal humanized IgG1 anti‑DPP4 mAb that seems to be well and/or cell surface pathways is facilitated by host pro- tolerated in patients with advanced solid tumours201. teases that cleave and activate S. Cathepsins are cysteine Koch’s postulates Criteria used to establish a For the treatment of SARS-CoV, small-molecule entry proteases that are involved in the endosomal path- causative relationship between inhibitors such as N-(2‑aminoethyl)-1‑aziridine- way and can be inhibited by cathepsin inhibitors such a microorganism and a disease. ethanamine (NAAE) inhibit the catalytic activity of as K11777 and its related vinylsulfone analogues111.

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These compounds seem to be safe and effective against associated with immunopathology during animal chal- various parasitic infections in animal models, and lenge. These are unfavourable approaches for MERS have broad-spectrum activities against enveloped vaccine development because a substantial proportion RNA viruses such as CoVs (SARS-CoV, MERS-CoV, of patients with severe MERS have comorbidities or HCoV‑229E and HCoV‑NL63), filoviruses (Ebola systemic immunocompromising conditions. Other and Marburg viruses) and paramyxoviruses111,204–206. vaccination strategies for MERS that use DNA plas- TMPRSS2 is a serine protease that mediates the cell mids, viral vectors, nanoparticles, virus-like particles surface entry pathway; camostat mesylate is a synthetic and recombinant protein subunits are in development low-molecular-weight serine protease inhibitor that and some have been evaluated in animal models157,216–233 has been used to treat chronic pancreatitis in humans (TABLE 4). The availability of a safe and effective MERS- with minimal side effects207,208. This molecule inhibits CoV vaccine for dromedary camels and non-immune SARS-CoV and MERS-CoV in vitro and improves sur- individuals at high risk of camel contact in endemic vival of SARS-CoV-infected mice109,111. Furin, another regions such as the Middle East and the greater Horn of ubiquitously expressed host protease, is also important Africa would be an important measure for controlling in MERS-CoV S-mediated entry. Blocking furin with the ongoing epidemic. decanoyl-Arg-Val-Lys-Arg-chloromethylketone inhibits MERS-CoV entry and cell–cell fusion in vitro110. Outlook and challenges Another group of candidate anti-CoV drugs Animal models for testing anti-CoV drugs. Suitable target the endocytosis of CoV during cell entry. animal models are especially important for testing Chlorpromazine is an antipsychotic drug used in the anti-CoV drugs because most of these drugs have not treatment of schizophrenia that also affects the assem- been used in humans. In contrast to the limited num- bly of clathrin-coated pits at the plasma membrane. ber of animal models established for the mild infections It is active against HCV, alphaviruses and numer- caused by HCoV‑229E, HCoV‑OC43, HCoV‑NL63 and ous CoVs, including SARS-CoV and MERS-CoV, HCoV‑HKU1, various small animal and non-human in vitro123. Cardiotonic steroids that bind sodium/ primate models have been evaluated for studies of the potassium-transporting ATPase subunit α1, such as pathogenesis and treatment of SARS and MERS234–237. ouabain and bufalin, also inhibit clathrin-mediated The identification of ACE2 and DPP4 as the functional endocytosis of MERS-CoV at nanomolar concentra- receptors for SARS-CoV and MERS-CoV, respectively, tions209. However, the use of these clathrin-mediated was essential to the development of animal models that endocytosis inhibitors in patients with CoV infections are representative of severe human disease101,102. A num-

is limited by either very high EC50/Cmax ratios or tox- ber of different non-human primates were found to be icity. Alternatively, endocytosis can also be suppressed permissive to SARS-CoV, but none of them consistently by a high pH. Chloroquine is an anti-malarial drug reproduced characteristics of the severe human dis- that sequesters protons into lysosomes to increase the ease, and mortality was not observed237. These models intracellular pH. It has broad-spectrum antiviral activ- were predominantly useful to fulfil Koch’s postulates238. ities against numerous CoVs (SARS-CoV, MERS-CoV, Small animals — including young and aged BALB/c HCoV‑229E and HCoV‑OC43) and other RNA viruses and C57BL/6 mice, knockout mice with deficiencies in in vitro123,210–214. However, it did not substantially reduce T, B and/or NK cells, golden Syrian hamsters and fer- viral replication in SARS-CoV-infected mice, possibly rets — could be productively infected with SARS-CoV, because the cell surface pathway was not simultaneously but few of them developed clinically apparent disease237. blocked215. The anti-CoV effects, pharmacokinetic and The best available small animal models for SARS uti- pharmacodynamic profiles and toxicity of the combi- lize transgenic mice that express human ACE2 and/or nations of different protease and endocytosis inhibitors mouse-adapted SARS-CoV strains that are capable of that target these different cell entry pathways should be causing lethal disease in mice239–241. The limited avail- further evaluated in vivo. ability of these ACE2‑transgenic mice and mouse- adapted virus strains remains a major obstacle to testing Development of MERS-CoV vaccines anti-SARS-CoV drugs. Rapid diagnostics and effective vaccines are often com- Similar to SARS, non-human primate models were plementary to antiviral treatment in the control of epi- also used to fulfil Koch’s postulates and investigate the demics caused by emerging viruses (BOX 1). Although pathogenesis of MERS. Rhesus macaques developed only there has not been any new human SARS case for over a mild, self-limiting disease and were not optimal for the 10 years, sporadic cases and clusters of MERS continue evaluation of treatments for MERS148,242,243. By contrast, to occur in the Middle East owing to the persistence MERS-CoV-infected common marmosets developed a of zoonotic sources in endemic areas, and these cases disseminated and fatal infection that closely resembled spread to other regions. Effective MERS-CoV vaccines severe human disease128,244. However, the availability of are essential for interrupting the chain of transmission common marmosets is limited and experiments on these from animal reservoirs and infected humans to suscep- small primates are technically demanding. Therefore, tible hosts. Live-attenuated vaccines, which have been other small animal models for MERS were evaluated. previously evaluated in animal models for SARS, might Unlike with SARS-CoV, most small animals — including be associated with disseminated infection in immuno- BALB/c mice, golden Syrian hamsters, ferrets and rabbits compromised patients. Inactivated vaccines might be — were not susceptible to MERS-CoV infection245–247.

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Box 1 | The complementary roles of novel rapid diagnostics and antiviral agents As demonstrated in the severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) epidemics, rapid and accurate laboratory diagnosis is essential for the clinical management and epidemiological control of coronavirus (CoV) infections. Real-time reverse transcription (RT)-PCR assays, which can quantify viral loads, have facilitated studies on viral shedding patterns and optimization of treatment and infection control strategies. The peak viral load in SARS was found to occur at day 10 after symptom onset and helped to predict the timing of clinical deterioration and the need for intensive supportive care18,249. Point‑of‑care nucleic amplification tests such as RT‑loop-mediated isothermal amplification and RT‑isothermal recombinase polymerase amplification are suitable for field evaluation, especially in resource-limited areas250,251. Similarly, assays that detect abundantly expressed CoV antigens, such as the nucleocapsid protein, can be used for fast and high-throughput laboratory diagnosis without requiring biosafety level 3 containment252,253. The rapid availability of complete genome sequences of most human and animal CoVs has minimized the time required for the design of new RT‑PCR assays, source identification and molecular surveillance for emerging CoVs254. This was well illustrated in the MERS epidemic, in which highly sensitive and specific RT‑PCR assays targeting unique gene regions such as the region upstream of the envelope (E) gene (upE region) were quickly developed after the complete genome sequence of MERS-CoV strains isolated from humans became available255,256. Comparative genomic studies quickly identified bats and camels carrying CoVs that were highly similar to MERS-CoV strains isolated from humans, and these two animals were determined to be the likely CoV reservoirs40,42–44,46,91. Continuous surveillance and analysis of MERS-CoV genomes obtained from patients and animals in different areas in the Middle East are important for detecting mutations that may increase the ability of the virus to be efficiently transmitted from person to person71. Data analyses from the sequencing of small RNAs and the use of locked nucleic acid probes have allowed the development of new assays that target short but abundantly expressed gene regions from CoV genomes, such as the leader sequences257. The increasing number of complete CoV genomes and diagnostic gene targets has enabled the development of multiplex assays that simultaneously detect multiple CoVs or multiple gene targets of a particular CoV257. The increasing use of these multiplex assays in clinical laboratories worldwide will enhance our understanding of the changing epidemiology of CoV infections and enable the stratification of at‑risk patients and contact groups for early treatment and prophylaxis. In addition to improving acute clinical diagnosis, diagnostic advances have facilitated other aspects of the control of CoV epidemics and anti-CoV drug development. The isolation of infectious virus particles from clinical specimens in cell culture has a limited role in the acute diagnosis of CoV infection, as most human-pathogenic CoVs are either difficult or dangerous to culture258. Nevertheless, recent advances have enhanced their use in CoV pathogenesis studies, which are important for identifying new treatment targets. The previously unculturable HCoV‑HKU1 can now be isolated from primary differentiated human tracheal bronchial epithelial cells and human alveolar type II cells that are cultured at an air–liquid interface259–262. Ex vivo organ culture enables the identification of important viral and host factors that are involved in the severe pulmonary and extrapulmonary manifestations of SARS and MERS263–267. The number of available human and animal cell lines from various organs is increasing, and these provide insights into tissue and species tropism258,268,269. Similarly, detection of specific anti-CoV antibodies in paired acute and convalescent sera samples are mainly useful for seroepidemiological studies and contact tracing, but not for acute diagnosis41,49,51,270. Novel assays such as the spike glycoprotein (S) pseudoparticle neutralization assay, which do not require biosafety level 3 containment, enable high-throughput antibody detection in large-scale seroepidemiological studies and outbreak investigations271.

Intranasal inoculation of adenoviral vectors expressing novel CoV. This is especially applicable to viral enzyme human DPP4 followed by MERS-CoV inoculation was inhibitors, mAbs and antiviral peptides that target S, as a novel method that rapidly rendered mice susceptible well as agents that target the host cell receptor. Second, to MERS-CoV infection, but the disease was relatively there are a limited number of animal models available mild and confined to the respiratory tract157. Transgenic for infections caused by HCoV‑229E, HCoV‑OC43, mice expressing human DPP4 develop severe pulmo- HCoV‑NL63 and HCoV‑HKU1. Even for SARS and nary and disseminated infection and are currently the MERS, experiments using suitable animal models such best available small animal model for MERS248. Potential as mice with transgenes encoding ACE2 or DPP4, and anti-MERS-CoV treatment options identified in in vitro non-human primates, are only available in a few des- antiviral assays should be further evaluated in these ignated research biosafety level 3 laboratories, and transgenic mice. these experiments are technically demanding. Last and most important, the mild clinical severity of infections Generic challenges in the clinical development of novel caused by HCoV‑229E, HCoV‑OC43, HCoV‑NL63 and anti-CoV drugs. There are a number of virological and HCoV‑HKU1, together with the absence of new SARS patient-associated factors that pose major challenges in cases, have made recruitment of patients into clinical the clinical development of novel anti-CoV drugs. First, trials difficult and reduced the incentives for pharma- CoVs are one of the most diverse and rapidly mutat- ceutical companies to develop specific antiviral drugs for ing groups of viruses, and novel CoVs emerge repeat- these CoV infections. The continuing threat of MERS- edly at unpredictable times. Therefore, most anti-CoV CoV to global health security 3 years after its first dis- drugs that specifically target the replication apparatus covery presents a golden opportunity to tackle current of an existing CoV may not be effective against another obstacles in the development of new anti-CoV drugs.

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Table 4 | MERS-CoV candidate vaccines in development Vaccine type Examples Vaccine design strategy Comments Refs

Live rMERS-CoV‑ΔE Deletion of the gene encoding • Attenuated SARS-CoV‑ΔE mutant virus induced protection in 218, attenuated MERS-CoV E rendered the mice and hamsters 287–295 virus mutant virus replication-compe‑ • No animal data are available for a rMERS-CoV‑ΔE‑based tent and propagation-defective vaccine yet • Risk of disseminated infection in immunocompromised patients

DNA plasmid MERS-CoV S DNA plasmids that encode • BALB/cJ mice vaccinated with MERS-CoV S-encoding DNA 219 DNA full-length MERS-CoV S developed neutralizing anti-MERS-CoV antibodies • The neutralizing antibody titre was boosted 10‑fold after vaccination with S1 protein • Rhesus macaques vaccinated sequentially with MERS-CoV S-encoding DNA and S1 protein had reduced CT scan abnormalities Viral vectors MVA-MERS‑S, Viral vectors (MVA or Ad) that • Both MVA and Ad vector-based vaccines induced neutralising 220–224, Ad5‑MERS‑S, express full-length MERS-CoV S anti-MERS-CoV antibodies in BALB/c mice 298 Ad5‑MERS‑S1, or the S1 subunit of MERS-CoV S • A MVA-MERS-S vaccine conferred mucosal immunity and Ad5‑S and induced serum neutralizing anti-MERS-CoV antibodies in Ad41‑S dromedary camels • Mucosal (intragastric) administration of Ad5‑S or Ad41‑S vaccines induced the production of antigen-specific IgG and neutralizing antibodies, but not antigen-specific T cell responses, in BALB/c mice • Systemic (intramuscular) administration of Ad5‑S or Ad41‑S vaccines induced antigen-specific neutralizing IgG antibodies, as well as T cell responses in splenic and pulmonary lymphocytes • Increased immunopathology with severe hepatitis in SARS-CoV-infected ferrets that were previously vaccinated with an MVA-based vaccine expressing full-length SARS-CoV S Nanoparticles MERS-CoV Purified MERS-CoV S-containing • BALB/c mice vaccinated with MERS-CoV or SARS-CoV 225 S-containing nanoparticles produced in insect S-containing nanoparticles developed neutralizing antibodies nanoparticles (Sf9) cells that were infected specific to the viral S with specific recombinant • Adjuvant matrix M1 or alum is required to elicit an optimal baculovirus containing the gene neutralizing antibody response encoding MERS-CoV S Virus-like VRP‑S VEE virus-like replicon particles • Vaccination of BALB/c mice transduced with Ad5‑hDPP4 with 157 particles containing MERS-CoV S VRP‑S reduced viral titres in lungs to nearly undetectable levels by day 1 after inoculation with MERS-CoV Recombinant S(RBD)‑Fc, Full-length MERS-CoV S or the • Vaccinated BALB/c mice and/or rabbits developed 226–233 protein S1(358–588)‑Fc, RBD subunit of MERS-CoV S neutralizing antibodies subunits S(377–588)‑Fc • Protective effects may be enhanced by combination with and rRBD different adjuvants • Non-neutralizing epitopes in full-length S-based vaccines may induce antibody-mediated disease enhancement Ad, adenovirus; CoV, coronavirus; CT, computerized tomography; E, envelope protein; hDPP4, human dipeptidyl peptidase 4; IgG, immunoglobulin G; MERS, Middle East respiratory syndrome; MVA, modified vaccinia virus Ankara; RBD, receptor-binding domain; rRBD, recombinant RBD; S, spike glycoprotein; SARS,severe acute respiratory syndrome; S(RBD)‑Fc, RBD of S fused to the antibody crystallizable fragment; S1(358–588)‑Fc, amino acid residues 358–588 of the S1 subunit of S fused to the antibody crystallizable fragment; VEE, Venezuelan equine encephalitis; VRP, virus replicon particle.

It is prudent that a well-organized, multidisciplinary, from proceeding beyond the in vitro stage. First, many

international collaborative network consisting of clini- drugs have high EC50/Cmax ratios at clinically relevant cians, virologists and drug developers, coupled to polit- dosages. Examples of such drugs include cyclosporine, ical commitment, is formed to carry out clinical trials chlorpromazine and interferon alfa. Second, some using anti-CoV drugs that have already been shown to have severe side effects or cause immunosuppression. be safe and effective in vitro and/or in animal models. For example, the use of high-dose ribavirin may be associated with haemolytic anaemia, neutropenia, Prioritization of virus-based and host-based treatment teratogenicity and cardiorespiratory distress. MERS- options for clinical development. Despite the report of a CoV-infected common marmosets treated with myco- large number of virus-based and host-based treatment phenolate mofetil developed a fatal infection with even options with potent in vitro activities for SARS and higher viral loads in their lungs and extrapulmonary MERS, only a few are likely to fulfil their potential in tissues than untreated controls did128. Agents target- the clinical setting in the foreseeable future. Most drugs ing host signalling pathways or receptors may induce have one or more major limitations that prevent them immunopathology. Furthermore, the lack of a reliable

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drug delivery method in vivo is particularly problem- Moreover, they are either marketed drugs (in the case atic for siRNAs and other agents that have not been of lopinavir–ritonavir and interferon beta‑1b) or they previously used in humans. have been successfully developed for other infections Looking ahead, the most feasible options that should (such as palivizumab, which is used for respiratory syn- be further evaluated in clinical trials for the ongoing cytial virus infection, and enfuvirtide, which is used for MERS epidemic include monotherapy or combinational HIV infection). In the long term, the development of therapies that include lopinavir–ritonavir, interferon novel, broad-spectrum, pan-CoV antiviral drugs that beta‑1b and/or mAbs and antiviral peptides targeting are active against a wide range of CoVs may become the MERS-CoV S. These agents have protective effects ultimate treatment strategy for circulating and emerging against MERS in non-human primate or mouse models. CoV infections.

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